Additives for animal feed

ABSTRACT

The present invention relates to the combination of organic acid salts with at least one active ingredient of plant origin, preferably essential oils, partially protected with vegetable fats and/or oils, which prevent these active ingredients from being digested by stomach enzymes during the digestive process. The sodium salts of short-chain acids, preferably salts of butyric, acid are preferred. The preferred essential oils are ginger, piperine, oregano, garlic, thymol, carvacrol, cinnamaldehyde and/or any combinations thereof. The combination of organic acid salts and the essential oils protected with vegetable fats and/or oils, are used as powerful promoters or stimulants for animal growth, as organic bactericides against pathogenic bacteria present in said animals and as modulators of immune response thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed under the provisions of 35 U.S.C. § 111(a) andis a continuation of U.S. patent application Ser. No. 13/980,469 filedon Oct. 3, 2013, and entitled “ADDITIVES FOR ANIMAL FEED” in the name ofEnrique PABLOS PÉREZ, et al., which claims the priority of InternationalPatent Application No. PCT/ES2012/070029 filed on 20 Jan. 2012, whichclaims priority of Spanish Patent Application No. 201130070 filed on 21Jan. 2011, all of which are hereby incorporated by reference herein intheir entirety.

FIELD OF THE INVENTION

The present invention refers to additives for animal feed based onorganic acid salts combined with active ingredients of plant origintogether coated with vegetable fats and/or oils. These organic acidsalts are used as promoters or stimulants of animal growth, modulatorsof the immune response and as bactericides. Therefore, the presentinvention is in the field of animal production and more specifically inthe fields of animal feed and health.

STATE OF THE ART

The trend in animal feed in Europe in recent years has been the searchfor a diet, which not only covers the feeding needs of the animals butalso reinforces their health and reduces pathological problems withoutusing medicines, to improve production and economic yields of thefarmer. One of the ways of controlling the health and immunity of theanimal is by means of controlling intestinal flora.

The use of additives has been common practice in animal feeding with theaim of improving production yields, improving health and thus achievingmore efficient utilisation of the food. Because of the prohibition ofthe use of antibiotics as growth promoting substances in the EuropeanUnion, (directive 1831/2003/CEE), additives have been sought that oftendemonstrate improved production compared even to the antibioticsthemselves (Dipeolu et al., 2005). Various studies carried out in recentyears have shown that organic acids are a good alternative. their modeof action lies, on the one hand, in reducing the pH in the stomach,which limits development of pathogens and helps in protein digestionand, on the other hand, in the ability of some of these organic acids toenter bacteria and block their metabolism. In this sense, butyric acid,also known as butanoic acid, has been studied for several years andbeneficial effects have been observed in several species: pigs, poultry,ruminants, etc. and even in fish. Together with acetic and propanoicacids, butyric acid belongs to the group of short-chain volatile fattyacids (VFA). It has been demonstrated that short-chain VFA can inhibitgrowth of the enterobacteriaceae bacterial group (Salmonella,Escherichia coli, etc.), (Van Immerseel et al. 2004). This inhibitionoccurs owing to the fact that the undissociated forms of the volatilefatty acids are able to cross bacterial membranes, interfere inbacterial metabolism and bring about their death. The acid anion alsointerferes with gene transcription of the bacteria, which prevents themfrom reproducing and causing infection. Butyric acid has a betterdiffusion coefficient than other VFAs, so it crosses the bacterial wallmore easily than other acids.

Along with the antibacterial effect, butyric acid has additional effectsthat make it unique: for example, it stimulates pancreatic secretion(Katoh and Tsudo, 1984; Sano et al, 1995), improves electrolyteabsorption, reduces the incidence of diarrhoea, increases microvilliregeneration in the intestine and increases their length (Galfi andBokori 1990, Lesson et al. 2005) while also increasing the area ofintestinal absorption giving rise to higher average weight of poultryand to better food conversion. Thus all these studies demonstrate thatbutyric acid is beneficial for the livestock farmer, both in terms ofnutrition and in terms of health.

It should be pointed out that the free form of butyric acid is difficultto handle because of its high corrosivity and volatility. to solve thisproblem, various presentations of butyric acid have been developed:salts of butyric acid, salts of microencapsulated butyric acid and saltsof butyric acid protected in a matrix of vegetable fats and/or oils,which protect part of the active ingredient and achieve slow release ofthis natural growth promoter, thus ensuring its growth potentiating andbactericidal action in the animal's digestive tract at the same time asproviding the highest protected concentration possible, to include theproduct in a range of slow-release natural growth promoters. Thisprotected acid salt is marketed under the name of Gustor BP-70 (norel,sa, spain); hereinafter in this invention it will be referred to asBP-70.

Partial protection of salts of butyric acid with vegetable oils and/orfats enables sodium butyrate to act effectively throughout the digestivetract of the animal, ensuring that the active ingredient reaches fromthe initial sections of the digestive tract to the most distal sections,not only acting as a natural growth promoter but also as a modulator ofthe immune response and a bactericide, reducing the possibility ofinfection by pathogenic bacteria. the effectiveness of Gustor BP-70 hasbeen demonstrated in areas where the salts of unprotected butyric aciddo not act (caecum/faeces) and in areas where these salts, inencapsulated form, have still not been able to act (crop), making it acomplete and effective product in the fight against bacterialinfections, especially against Salmonella enteriditis, in bird nutrition(Fernández-Rubio c et al, 2009).

The function of sodium butyrate as a bactericide has been mainlyemployed in the treatment of Salmonella enteritis, as this is one of themain pathogenic bacteria affecting animals and man. Among the pathogenicbacteria is the bacterium Clostridium perfringens, which causes necroticenteritis as a consequence of the production of various toxins,resulting in extensive necrosis of the intestinal mucosa, mainlyaffecting chickens, turkeys, ducks and wild birds. C. perfringens occursnaturally in the intestine, proliferating under certain circumstancessuch as, for example, excess undegraded and undigested nutrients thatappear more frequently with dietary changes, especially when animalsreceive poorly balanced diets or containing poor quality raw materials,poor immune response of the gut-associated lymphoid tissue (GALT) due toexcess wear of the intestinal epithelium as a consequence of milling thefeed, presence of mycotoxins, coccidia, etc., unbalancing of theintestinal microbial flora and other conditions. In 1972, with therelease on the market of the coccidiostat Monensin, the appearance ofcases of infection by C. perfringens appeared to stop but with the trendfor removing antibiotics as growth promoters and for the addition ofcoccidiostats to the feed, there is a growing trend in the reappearanceof this disease.

Therefore, to resolve this growing problem of the rise of infectiousdiseases in animals for fattening to be used for human consumption, andalso to improve the growth of these animals, the present invention hasdeveloped a new additive for animal feed comprising salts of organicacids, preferably butyric acid, with active ingredients of naturalorigin, preferably essential oils, together partially coated withvegetable fats and/or oils. This additive is able to inhibit bacterialgrowth, especially of Gram-positive bacteria and coccidia, andadditionally improves the condition of the intestinal epithelium andimproves the weight of treated animals. Partial coating of the activeingredients of this feed additive for animals, the salt of an organicacid and essential oils, with fats and/or oils of plant origin protectsthese active ingredients from digestion in the stomach, but enables themto be active throughout the gastrointestinal tract of animals becausethrough this partial coating of the active ingredients, phases of theproduct with various amounts of fat (0%-50% fat) can be produced thatare digested and therefore released at different points of thegastrointestinal tract of the animals: the part unprotected by fat isreleased and has effect in the first sections of the gastrointestinaltract, up to the small intestine; the part protected by fat is notreleased until the fat starts to be digested by the action of pancreaticlipases. Given the slow digestion of the fat, in vitro studies haveshown that part of the active ingredient can reach the most distal partsof the gastrointestinal tract, exercising its action there.

The feed additive described in the present invention is also able topotentiate the development of the intestinal epithelium, encouraging thegrowth of intestinal villi, which as a consequence of the necroticenteritis induced in the animals described in the examples of thepresent invention are reduced in number and size. Furthermore, theincrease in development of the intestinal epithelium encourages improvedabsorption of food, thereby causing an increase in growth and fatteningof the animals feeding with the additive. The feed additive described inthe present invention is also able to reduce the incidence of bacterialdiseases such as necrotic enteritis or those caused by Salmonellaenteriditis, owing to the modulating action of the immune response andits bactericidal action against pathogens including C. perfringens andS. enterica. The combined use of the organic acid salt and essentialvegetable oils together partially coated with vegetable fats and/or oilsis able to potentiate the physiological promoter effect of butyrate suchas, for example as mentioned above, in the development of intestinalvilli, in addition to potentiating its bactericidal effect.

The other preferred active ingredients, to be included in thecombination with organic acid salts, are essential oils. The mainsynergies of essential oils with organic acid salts from a nutritionalpoint of view are their action as stimulators of digestibility, becausethey encourage the balance and control of microbial flora; they are alsoimportant for their action as stimulators of immunity and antimicrobialand antioxidant properties.

Furthermore, in the present invention, the use of the combination ofthese organic acid salts and essential oils, together protected withoils and/or fats of plant origin, as a feed additive for animals isdescribed, this feed additive acting as a natural growth promoter aswell as bactericide and immunomodulator, able to reduce levels ofpathogenic bacteria, specifically C. perfringens and S. enterica, andimproving the immune response with which animals are protected frombacterial infections.

DESCRIPTION OF THE INVENTION Brief Description of the Invention

The present invention describes a new additive for animal feed that actsas a natural promoter of animal growth and comprises organic acid saltscombined with active ingredients of natural origin, preferably essentialoils. This combination is partially protected from digestion in thestomach by a partial coating of vegetable fats and/or oils. Thiscoating, being partial, does not prevent the action of the mentionedactive ingredients throughout the whole intestinal tract, event to themost distal parts.

The term growth promoters or stimulators is given to those additivesthat form an integral part of animal diet and that perform the functionof improving the daily weight gain of animals (DWG), as well asconversion of the ration consumed. In the present invention, growthpromoters or stimulators can be administered by different ways, eitherby injection, in implants or as an additive in the feed of the animals.In the present invention, the effect of growth is measured as anincrease in weight.

For the purpose of the present invention, the term organic acid refersto compounds containing one or more carboxylic groups (—COOH) in theirformula, proton donors that can have various functional groups such ashydroxyacids, ketoacids, aromatic acids, heterocyclic compounds and alsoamides and lactones. The organic acids used in the present invention arepreferably volatile fatty acids, preferably short-chain, and can beselected from: butyric acid, propionic acid, formic acid, lactic acid,citric acid, lauric acid, capric acid, caprylic acid, caproic acid,acetic acid and others. The proportions of the organic acid in theadditive for animal feed described in the present invention may varybetween 30% and 70% of the wet weight of the total product; preferablythe proportion is 50%.

The salts of the organic acids used in the present invention arepreferably the sodium salt, cupric salt, potassium salt, calcium saltand others. The proportions of the organic acid salt in the additive foranimal feed described in the present invention may vary between 30% and70% of the wet weight of the total product; preferably the proportion is60%.

In the present invention, the use of the sodium salt of butyric acid(sodium butyrate) is preferred, although any of the salts of the acidslisted above may be used.

For the purpose of the present invention, the term active ingredient ofplant origin refers to any material of plant origin to which appropriateactivity can be attributed that is capable of exercising a beneficialeffect on the organism to which it is applied. The active ingredients ofplant origin used in the present invention are preferably essentialoils. For the purposes of the present invention, the term essential oilrefers to a volatile organic substance or substances belonging tovarious classes of compounds, for example hydrocarbons, esters,alcohols, aldehydes, some acids, phenols and their derivatives,lactones, etc., all long-chain products from plant biosynthesis, calledplant secondary metabolites. The term essential oil is also applied tosimilar synthetic substances, prepared from coal tar, and semi-syntheticsubstances prepared from the natural essential oils. The essential oilsused in the present invention can be selected from any of the followinglist: ginger, piperine, oregano, garlic, thymol, carvacrol,cinnamaldehyde, etc. The proportions of active ingredients of plantorigin in the additive for animal feed described in the presentinvention may vary between 1% and 20% of the wet weight of the totalproduct, preferably the proportion is 10% of the final product.

For the purposes of the present invention, the term vegetable or plantfat and/or oil refers to an organic compound obtained from seeds orother parts of plants that accumulate in the tissues as an energysource. The vegetable fats and/or oils used in the present invention tocoat the combination of the organic acid salt and the active ingredientsof natural origin, preferably essential oils, can be selected from: palmstearin (the more solid part obtained after fractioning palm oil) andcalcium soap of palm oil fatty acids (palm fatty acid distillate, PFAD)(saponification with calcium of the fatty acids). The proportions ofthese vegetable fats and/or oils coating the organic acid salt andessential oils described in the present invention may vary between 30%and 60% of the wet weight of the total product, preferably theproportion is 30%.

Furthermore, the present invention describes the use of this additive,natural animal growth promoter, in addition to improving productiveparameters of animals (for example increasing their weight), as animmune response modulator and as a bactericide, being able to reduce theincidence in these animals of bacterial diseases such as, for example,necrotic enteritis in poultry, and may also be used for other types ofbacterial diseases such as, for example: Salmonella enteritis,Escherichia coli, Campylobacter spp., etc. and in other animals such as,for example: rabbits, pigs, etc.

For the purposes of the present invention, the term bactericide isdefined as any product, agent or substance able to remove of killbacteria. For the purposes of the present invention, the bactericidaleffect or action is measured as macroscopic changes produced in theintestines of animals due to bacterial infection. Also, for the purposesof the present invention, modulation of the immune response is measuredas the change in gene expression with respect to control animals notreceiving the additive of the invention in their feed, infected or not,of genes that code for the cytokines, preferably IL-1β, IL-2, CD3δγ andTNFSF15.

DESCRIPTION OF THE FIGURES

FIG. 1. Weights of animals of the groups A=control and B=BP-70+gingerand piperine on day 17 of the experimental trial. The Y axis shows theweight in grams. The X axis shows the time in days. * indicatesstatistically significant differences compared to the infected butuntreated control group (p<0.05). BP-70: sodium butyrate (60% of the wetweight) protected with plant oil (30% of the wet weight). The percentageof ginger and piperine included in the additive was 10% of the wetweight of the total product of each of the essential oils that is 5%ginger and 5% piperine.

FIG. 2. Weights of animals of the groups A=control and B=BP-70+gingerand piperine on day 24 of the experimental trial. The Y axis shows theweight in grams. The X axis shows the time in days. * indicatesstatistically significant differences compared to the infected butuntreated control group (p<0.05). BP-70: sodium butyrate (60% of the wetweight) protected with plant oil (30% of the wet weight). The percentageof ginger and piperine included in the additive was 10% of the wetweight of the total product of each of the essential oils, that is 5%ginger and 5% piperine.

FIG. 3. Comparison of the macroscopic changes in samples of intestine ofthe animal group treated with BP-70+ginger and piperine additives (GroupB) compared to the macroscopic changes shown by the infected butuntreated control animals (Group A). The Y axis shows the score of themacroscopic changes of each group. * indicates statistically significantdifferences compared to the control group (p<0.05). BP-70: sodiumbutyrate (60% of the wet weight) protected with plant oil (30% of thewet weight). The percentage of ginger and piperine included in theadditive was 10% of the wet weight of the total product, that is 5%ginger and 5% piperine.

FIG. 4A shows the relative levels of expression of the genes IL-1β,IL-2, IL-8, IL-10, CD3γδ, LITAF and RNFSF15 in the groups of animalstreated with BP-70 (Group C) compared to the uninfected and untreatedanimal group (Group A). BP-70: sodium butyrate (70% of the wet weight)protected with plant oil (30% of the wet weight). The squared area ofthe box spans 50% of all measurements, the horizontal line inside thebox represents the mean of the sample and the vertical lines represent50% of the measurements that were outside the values of the box. Theasterisks indicate statistical significance with a value of p<0.05. Therelative levels of gene expression indicate the expression of each genecompared to the levels of expression of each gene in Group A.

FIG. 4B shows the relative levels of expression of the genes IL-1β,IL-2, IL-8, IL-10, CD3γδ, LITAF and RNFSF15 in the groups of animalstreated with BP-70 or BP-70+ginger (Group D) compared to the uninfectedand untreated animal group (Group A). BP-70+ginger+piperine: sodiumbutyrate (60% of the wet weight) protected with plant fat (30% of thewet weight)+5% ginger+5% piperine. The squared area of the box spans 50%of all measurements, the horizontal line inside the box represents themean of the sample and the vertical lines represent 50% of themeasurements that were outside the values of the box. The asterisksindicate statistical significance with a value of p<0.05. The relativelevels of gene expression indicate the expression of each gene comparedto the levels of expression of each gene in Group A.

FIG. 4C shows the relative levels of expression of the genes IL-1β,IL-2, IL-8, IL-10, CD3γδ, LITAF and RNFSF15 in the groups of animalstreated with BP-70 or BP-70+ginger (Group F) compared to the uninfectedand untreated animal group (Group A). BP-70+ginger+piperine: sodiumbutyrate (60% of the wet weight) protected with plant fat (30% of thewet weight)+5% ginger+5% piperine. The squared area of the box spans 50%of all measurements, the horizontal line inside the box represents themean of the sample and the vertical lines represent 50% of themeasurements that were outside the values of the box. The asterisksindicate statistical significance with a value of p<0.05. The relativelevels of gene expression indicate the expression of each gene comparedto the levels of expression of each gene in Group A.

FIG. 5. Relative levels of expression (Y axis) of the gene TNFSF15 inthe various treatment groups: BP-70 (Groups C and E) orBP-70+ginger+piperine (Groups D and F), compared to untreated anduninfected animals (Group A). BP-70: sodium butyrate (70% of the wetweight) protected with vegetable oil (30% of the wet weight).BP-70+ginger+piperine: sodium butyrate (60% of the wet weight) protectedwith plant fat (30% of the wet weight)+5% ginger+5% piperine. Group B:infected animals but not treated; Group C: uninfected animals treatedwith BP-70: Group D: uninfected animals treated withBP-70+ginger+piperine; Group E: infected animals treated with BP-70;Group F: infected animals treated with BP-70+ginger+piperine. Thesquared area of the box spans 50% of all measurements, the horizontalline inside the box represents the mean of the sample and the verticallines represent 50% of the measurements that were outside the values ofthe box. The asterisks indicate statistical significance with a value ofp<0.05. The relative levels of gene expression indicate the expressionof each gene compared to the levels of expression of each gene in groupa.

FIG. 6. Relative levels of expression (Y axis) of the genes IL-1β, IL-2,IL-8, IL-10, CD3γδ, LITAF and RNFSF15 in the group of animals infectedand treated with BP-70+ginger+piperine (Group F) compared to infectedand untreated animals (Group B). BP-70+ginger and piperine: sodiumbutyrate (60% of the wet weight) protected with vegetable fat (30% ofthe wet weight)+5% ginger+5% piperine. The squared area of the box spans50% of all measurements, the horizontal line inside the box representsthe mean of the sample and the vertical lines represent 50% of themeasurements that were outside the values of the box. The asterisksindicate statistical significance with a value of p<0.05. The relativelevels of gene expression indicate the expression of each gene comparedto the levels of expression of each gene in Group B.

FIG. 7. Relative levels of expression (Y axis) of the gene TNFSF15 inthe groups of animals infected and treated with BP-70 (Group E) or withBP-70+ginger+piperine (Group F) compared to infected and untreatedanimals (Group B). BP-70: sodium butyrate (70% of the wet weight)protected with plant oil (30% of the wet weight). BP-70+ginger+piperine:sodium butyrate (60% of the wet weight) protected with plant fat (30% ofthe wet weight)+5% ginger+5% piperine. The squared area of the box spans50% of all measurements, the horizontal line inside the box representsthe mean of the sample and the vertical lines represent 50% of themeasurements that were outside the values of the box. The asterisksindicate statistical significance with a value of p<0.05. The relativelevels of gene expression indicate the expression of each gene comparedto the levels of expression of each gene in Group B.

DETAILED DESCRIPTION OF THE INVENTION

One of the objects of the present invention refers to additives foranimal feed comprising the combination of organic acid salts with atleast one active ingredient of plant origin both together partiallycoated with vegetable oils and/or fats.

In a preferred embodiment, the additives of the invention arecharacterised in that the organic acids are selected from any of thelist: butyric, lactic, citric, lauric, capric, caprylic, caproic, aceticand others, with butyric acid being preferred.

In another preferred embodiment, the additives of the invention arecharacterised in that the organic acid salts are selected from any ofthe list: sodium, calcium, cupric and potassium. The proportions of theorganic acid salt in the additive for animal feed described in thepresent invention may vary between 30% and 70% of the wet weight of thefinal product, preferably the proportion is 60%.

In another preferred embodiment, the additives of the invention arecharacterised in that the preferred salt is the sodium salt and thepreferred organic acid is butyric acid; therefore the sodium salt ofbutyric acid is in the preferred additive of the invention.

In another preferred embodiment, the additives of the invention arecharacterised in that the salt of the organic acid is preferably at aconcentration of 60% of the wet weight of the final product.

In another preferred embodiment, the additives of the invention arecharacterised in that the active ingredients of plant origin arepreferably essential oils and can be selected from any included in thelist: ginger, piperine, oregano, thymol, garlic, carvacrol,cinnamaldehyde and/or any of their combinations, preferably thecombination of the essential oils is ginger and piperine.

In another preferred embodiment, the additives of the invention arecharacterised in that the vegetable active ingredients are preferably ina proportion of between 1% and 20% of the wet weight of the finalproduct, preferably the proportion is 10% of the wet weight of the finalproduct.

In another preferred embodiment, the additives of the invention arecharacterised in that the combination of essential oils of ginger andpiperine is preferably 5% ginger oil and 5% piperine oil.

In another preferred embodiment, the additives of the invention arecharacterised in that the partial coating of the combination of organicacid salts and active ingredients of plant origin with vegetable oilsand/or fats is approximately between 30% and 60% of the wet weight ofthe final product, preferably the coating is 30%.

In another preferred embodiment, the additives of the invention arecharacterised in that the vegetable oils and/or fats that partially coatthe combination of organic acid salts and active ingredients of plantorigin are selected from: palm stearin and the calcium soap of palmfatty acid distillate (PFAD), preferably palm stearin.

In another preferred embodiment, the additives of the invention arecharacterised in that the salt is preferably the sodium salt of organicbutyric acid, the active ingredient of plant origin is preferablycomposed of the combination of essential oils, preferably the essentialoils of ginger and piperine and the coating of vegetable oils and/orfats is preferably palm stearin.

In another preferred embodiment, the additives of the invention arecharacterised in that the concentration of sodium butyrate is preferably60%, the concentration of the combination of the essential oils ofginger and piperine is preferably 10% and the coating of palm stearin ispreferably 30%.

In another preferred embodiment, the combination of essential oils ofginger and piperine is preferably composed of 5% ginger oil and 5%piperine oil.

Another of the objects described in the present invention refers to theuse of the additives described above for animal feed, as promoters ofanimal growth, preferably of chickens, rabbits and pigs.

Another of the objects described in the present invention refers to theuse, in animal feed for breeding animals fed with the additivesdescribed above, as modulator of the immune response. In anotherpreferred embodiment, the modulation of the immune response comprises achange in the expression of any of the genes that code for any of thefollowing cytokines: increasing the expression of IL-1β, IL-2, CD3γδ,and/or reducing the expression of TNFSF15.

Another of the objects described in the present invention refers to theuse, in animal feed for breeding animals fed with the additivesdescribed above, as a bactericide, inhibiting the growth of bothGram-positive bacteria, preferably bacteria of the genus clostridium,more preferably C. perfringens, Gram-negative bacteria, preferablybacteria of the Enterobacteriaceae family, more preferably of the generasalmonella, Escherichia and campylobacter including protozoans such asthe case of the protozoans of the Eimeria and Cryptosporidium families.

The purpose of the examples that are described below is to illustratethe present invention but without limiting its scope.

Example 1. Macroscopic and Histological Pathological Examination inBroiler Chickens that were Induced with Necrotic Enteritis and Treatedwith Various Feed Additives

Conventional broiler chickens were used (hybrid Babolna, Babolna,Hungary) that were one day of age when they were distributed to thevarious treatment groups described in the present invention. Althoughthe examples were carried out in chickens for fattening (poultryfarming), the feed additives that potentiate animal growth and withnatural bactericidal action, described in the present invention, can beused in any other type of farming such as, for example: pig, rabbit,sheep, cow, etc.

The broiler chickens were fed a conventional diet free of medicines andrich in proteins (25% fishmeal). This feed had no coccidiostats (agentsuseful in the treatment or prevention of coccidiosis in humans and/oranimals). The animals had no restriction to water and were kept in cagesat a density of 30 animals/m². The bedding, watering and feedingequipment were previously sterilised in an autoclave. The ambienttemperature was initially set at 32±4° C., reducing to 24±4° C. as theanimals grew.

To induce necrotic enteritis, these animals were inoculated with thealpha-toxigenic type a strain of C. perfringens (ATCC 13124). Thisstrain was gown in Reinforced Clostridial Medium (BD, MD, USA) culturemedium at a temperature of 37° C. for 24 hours in anaerobic conditionsmaking use of the Anaerocult A (Merck, Darmstadt, Germany) system. Thenthese bacterial cultures were centrifuged (3000 g, 10 min., 5° C.,Universal centrifuge 320R, Hettich centrifuges) and the pellet obtainedwas resuspended in a solution of sterile phosphate buffered saline(PBS). The C. perfringens titre was 3-4×10⁸ colony forming units/ml(CFU/ml). Necrotic enteritis only shows clinical signs if predispositionfactors are present. Therefore these animals were infected orally withcoccidia by vaccination with the live attenuated vaccine Paracox 5®(Ceva-Phylaxia) and with C. perfringens A.

Table 1 shows the infection protocol and vaccination used in the presentinvention. Briefly, the animals were vaccinated against Gumboro diseaseor infectious bursitis disease (IBD). This is a highly contagiousdisease in young chickens caused by infectious bursitis disease virus(IBDV) and is characterised by immunosuppression and death of theanimals, usually at the age of between 3 to 6 weeks of age. The vaccineagainst this disease (CEVAC gumbo L®, Ceva-Phylaxia) was administered inwater on day 16 to cause a moderate immunosuppression and to induce theanimals to be more susceptible to suffering from necrotic enteritis.Next, they were inoculated orally through a nasogastric tube with 2 mLof a suspension of C. perfringens (6.8×10⁸ CFU) three times a day (8:00,12:00 and 16:00) on days 18, 19, 20 and 21. On day 19, they were alsogiven a dose 10 times higher than normal of the attenuated live vaccineParacox-5® (Ceva-Phylaxia), to induce infection by coccidia and topredispose the animals to the clinical disease. The Paracox-5® vaccineis an attenuated live vaccine containing sporulated oocysts derived fromprecocious strains of coccidia that are pathogenic for chickens: Eimeriaacervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimerianecatrix, Eimeria praecox and Eimeria tenella. Control animals were alsotreated with vaccines against gumboro disease and with the Paracox-5®vaccine.

TABLE 1 Infection and vaccination protocol Age of chickens (days) 1 1617 18 19 20 21 Vaccination against Gumboro x Infection with Clostridiumx x x x Vaccination with Paracox-5 ® x (10 times higher dose thannormal)

The broiler chickens (n=24) in this present example were divided intothree groups (12 animals/group, Table 2). All the birds were treatedwith vaccines against Gumboro disease and with the Paracox-5® vaccine onthe days indicated in Table 1. All the animals of each group were orallyinfected with C. perfringens A (6-8×10⁸ CFU). The control group ofanimals were fed the base diet described, without any additive. Incontrast, the other group of animals were fed a diet containing aconcentration of 1.5 g/kg feed of the additive described in the presentinvention: sodium butyrate with ginger and piperine, both activeingredients coated in 30% wet weight of vegetable fats, specificallypalm stearin. The sodium butyrate used was the composition from Norel,S.A. called BP-70, to which was added essential oils of ginger (CAS84696-15-1) (5% wet weight) obtained from Ventós S.A. (Barcelona, Spain)and piperine (5% wet weight) obtained from the company SensientFragances S.A. (Granada, Spain). BP-70 is composed of 70% of the wetweight of sodium butyrate and 30% of the wet weight of vegetable fats.The 30% of vegetable fats protects approximately 43% of the total sodiumbutyrate, therefore the product BP-70 can be described as being composedof 30% protected sodium butyrate and 40% unprotected sodium butyrate.The essential oils of ginger and piperine were coated to the samepercentages as the sodium butyrate. All the additives for the feed weresupplied by Norel, SA, Spain. The body weight of the animals wasmeasured during the experiment on days 1, 4, 7, 10, 14, 18 and 21. Allthe animals were sacrificed on day 25.

TABLE 2 Experimental design. BP-70 + ginger + C. Animal piperineperfringens groups (g/kg feed) Paracox-5 ® Gumboro A A (n = 12) — + + +B (n = 12) 1.5 g/kg + + + Grupo A = control, Grupo B = BP-70 + ginger +piperine, Group C = essential oils.

FIG. 1 shows that already on day 17, the day before infection with C.perfringens A, the group of animals treated with BP-70+ginger+piperine(Group B) showed an increase in body weight compared to the infectedcontrol group but not treated with any feed additive (Group A)(p=0.002). Then another body weight analysis was conducted for each ofthe groups of animals in the study on day 24, one day before theirsacrifice. FIG. 2 shows that similarly to the body weight analysis onday 17 (FIG. 1), this analysis demonstrated that the group treated withBP-70+ginger+piperine showed a significantly higher body weight gainthan the control animal group that were infected but not treated withany feed additive (p=0.001).

Next, a histopathological analysis was conducted of the samplescollected from various organs of each group of animals: liver, spleenand distal jejunum. Fresh samples were immediately submerged in 10%formalin for preservation until they were used in subsequent analyses.Microscopic lesions were classified in accordance with the method ofGholamiandehkordi et al. (2007) with small deviations: 0=negative;1=inflammation of the intestine; 2=focal necrosis (1.2 cm) ordegenerative changes in the mucosa (fatty appearance); 3=irregularnecrosis (3-4 cm) and 4=diffuse necrosis. The presence of enteritis(generally histiocytic lymphoma) was also noted.

FIG. 3 shows a comparison of the severity of the macroscopic lesionsseen in the group of animals treated with BP-70+ginger+piperine (GroupB) compared to the control group of animals infected but not treatedwith any additive. As shown in FIG. 3, Group B (BP-70+ginger+piperine)showed significantly lower scores than the control group (p=0.001).Therefore, these results demonstrate that treatment withBP-70+ginger+piperine (Group B) significantly reduced macroscopiclegions found in broiler chickens infected with C. perfringens Acompared to control animals that showed infection but were notadministrated with any type of additive (Group A).

Thus the present example demonstrates that treatment with the feedadditive described in the present invention, sodium butyrate and plantessential oils, ginger and piperine, together partially coated withvegetable fats, produces a significant increase in the weight of theanimals with artificial necrotic enteritis induced by inoculation withC. perfringens A and treated with this additive (Group B) compared tothe control animal group, which also showed signs of infection but werenot treated with this additive (Group A). This example also demonstratesthe improvement in the lesions, mainly intestinal (caused by infectionwith C. perfringens A), that the samples extracted from the group ofanimals treated with the additive of the invention (Group B) showedcompared to the control animal group that were not treated with anyadditive (Group A).

Example 2. Determination of the Immune Response of Gut-associatedLymphoid Tissue (GALT) in Broiler Chickens Suffering from NecroticEnteritis and Treated with Various Feed Additives

In order to determine the immune response of the GALT, anotherexperimental trial was carried out with broiler chickens (n=48) thatwere divided into six groups (6 animals/group, Table 3). All the birds,similarly to Example 1, were treated with vaccines of Gumboro andParacox-5® on all the days indicated in Table 1. The groups A, C and Dwere composed of broiler chickens not infected with C. perfringens A,whereas groups B, E and F were composed of broiler chickens orallyinfected with C. perfringens A (6-8×10⁸ CFU). The base diet of theanimals was the same as described in Example 1, but complemented withvarious additives for feed: (i) 1.5 g of BP-70/kg. of feed (Groups C andE), (ii) 1.5 g of BP-70+ginger+piperine/kg feed (Groups D and F). Allthe additives for feed were supplied by Norel, SA (Madrid, Spain).

TABLE 3 Experimental design BP-70 + BP-70 ginger + Paracox- Clostridium(g/kg piperine: 5 ® Gumboro perfringens Group feed) (g/kg feed) vaccinevaccine A A — — + + − B — — + + + C 1.5 g/kg — + + − D — 1.5 g/kg + + −E 1.5 g/kg — + + + F — 1.5 g/kg + + +

The chickens were sacrificed on day 22 and intestinal samples were takenof approximately 5 cm length of the jejunum between Meckel'sdiverticulum and the iliac region. The intestinal sections were cutlongitudinally and were washed three times with iced PBS containing 100U/ml penicillin and 100 μg/ml streptomycin (Fischer Scientific). Themucous layer was scraped with a sterile glass slide and the intestinaltissue was immediately submerged in 1 ml ice cold TRizol (Invitrogen)for subsequent extraction of RNA according to the manufacturersinstructions. The samples were homogenised and maintained at −80° C.until processing.

In order to evaluate the inflammatory and immune response after inducinginfection with necrotic enteritis in the animals and comparing theeffects of each of the treatments with the various additives describedin Table 3, the changes in the levels of expression of various cytokineswas evaluated by the technique of quantitative PCR (qRT-PCT). Thecytokines analysed were: interleukin-1 beta (IL-1β), interleukin-2(IL-2), interleukin-8 (IL-8), interleukin-10 (IL-10), CD3γδ receptor,lipopolysaccharide-induced tumour necrosis factor-alpha factor (LITAF)and factor 15 of the super-family of the tumour necrosis factor(TNFSF15). The control genes used as reference were:glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin. Theprimers used for each of the genes mentioned are shown in Table 4. Theinterleukins-1, 2, 8 and 10 are members of the interleukin family,essential for the cellular and humoral immune response. The CD3 receptor(cluster of differentiation 3) is a target receptor found in allT-cells, LITAF is a cytokine of the TNF family that is released afterLPS induction and TNFSF15 is present in endothelial cells and alsoparticipates in proliferation, differentiation and apoptosis of thecells of the immune system.

In order to analyse significant differences shown by each of thetreatment groups described above in the levels of expression of thegenes analysed, the ANOVA test was used.

TABLE 4 Primers used in the qRT-PCT technique Product RNA Primers sizeGenBank Gene Direct Inverse (bp) Accession No. GAPDH SEQ ID No. 1 SEQ IDNo. 2 264 NM_204305 β-actin SEQ ID No. 3 SEQ ID No. 4 169 NM_205518IL-1β SEQ ID No. 5 SEQ ID No. 6 80 NM_204524 IL-2 SEQ ID No. 7 SEQ IDNo. 8 148 NM_204153 IL-8 SEQ ID No. 9 SEQ ID No. 10 200 NM_205498 IL-10SEQ ID No. 11 SEQ ID No. 12 94 NM_001004414 CD3γδ SEQ ID No. 13 SEQ IDNo. 14 166 NM_205512.1 LITAF SEQ ID No. 15 SEQ ID No. 16 229 NM_204267TNFSF15 SEQ ID No. 17 SEQ ID No. 18 292 NM_001024578

In order to demonstrate the immunomodulator effects provided by the feedadditives administered to the various animal groups described in thepresent example, either infected or not with C. perfringens, animalGroups C and D that were treated with some of the additives mentionedand that did not show infection were compared to the untreated anduninfected control group of animals (Group A). Also, the animal groups Eand F that were treated with the additives BP-70 orBP-70)+ginger+piperine respectively and that showed necrotic enteritiswere compared against the group of animals infected with C. perfringensbut not treated with any additive of those described in the presentinvention (Group B).

Firstly, the levels of expression of the cytokines analysed in theuntreated control group of animals that were not infected with C.perfringens A (Group A) were compared to the groups of animals notinfected with C. perfringens A but treated with the feed additives BP-70(Group C) and BP-70+ginger+piperine (Group D) to analyse the naturalimmunomodulator effects of these additives.

FIGS. 4A, B and C show the immunomodulator effect of the additives:BP-70 and BP-70+ginger+piperine on the level of expression of the genesIL-1β, IL-2, IL-8, IL-10, CD3γδ, LITAF and TNFSF15. The relativeexpression ratios of these genes in the treated groups C and E werecompared to those in untreated animals (Group A). None of the groupswere infected with C. perfringens A. FIG. 4A shows that treatment withBP-70 (Group C) did not cause any change in the expression levels of thegenes analysed. The major changes in the expression profile of the genesanalysed occurred due to the treatment with the additiveBP-70+ginger+piperine (Group D) (FIG. 4B). Three of the genesinvestigated were over expressed in the group of animals takingBP-70+ginger+piperine (Group D) compared to the untreated control groupof animals (Group A). These genes were: IL-1β (p=0.028), IL-2 (p=0.026)and CD3γδ (p=0.011). One of the genes analysed, TNFSF15, showedunderexpression (p=0.004). This underexpression was also reproduced(FIG. 4C) when comparing the untreated and uninfected control group ofanimals (Group A) to the infected group of animals treated with theadditive described in the present invention, BP-70+ginger+piperine(Group F). Overexpression of the genes IL-1β and IL-2 indicates theactivation of the immune response cascade, giving rise to stimulation ofproliferation and maturation of lymphocytes. These observations wereconfirmed by the increase in gene expression of CD3γδ that represents araised total T-cell count.

FIG. 5 also shows the levels of expression of each of the cytokinesanalysed: IL-1β, IL-2, IL-8, IL-10, CD3γδ, LITAF and TNFSF15, in each ofthe groups of animals treated with each of the additives described inthe present example and that were either infected or not with C.perfringens A (Groups C to F), as well as the control group of animalsinfected with C. perfringens A, but not treated with any of theseadditives (Group B). FIG. 5 demonstrates that treatment withBP-70+ginger+piperine (Group E) caused underexpression of the geneTNFSF15 compared to both the control and uninfected group of animalsthat were not given any additive in their diet (Group A) and in theinfected group of animals that were also not given any additive in theirdiet (Group B).

Secondly, the immunomodulator effects provided by feed additives givento animals of the groups E (BP-70) and F (BP-70+ginger+piperine),treated with the additives mentioned and that had necrotic enteritis,were analysed with respect to the group of animals infected with C.perfringens but not treated with any additive of those described in thepresent invention (Group B).

FIG. 6 shows the levels of expression of the genes IL-1β, IL-2, IL-8,IL-10, CD3γδ, LITAF and TNFSF15 in the group of animals infected with C.perfringens A and that were treated with the feed additive described inthe present invention, BP-70+ginger+piperine (Group F), compared to thelevels of expression of these genes in the group of control animalsinfected with C. perfringens A but that were not fed with the additivesused in the present example (Group B). This analysis was carried out toanalyse the effects of these additives in the evolution of theinfection. FIG. 6 shows that there was a significant increase in thelevels of expression of cytokine IL-2 compared to the control untreatedgroup (Group B). Furthermore, the group of animals treated withBP-70+ginger+piperine showed a decrease in the levels of expression ofTNFSF15 compared to the untreated control group of animals (Group B).

Similarly, FIG. 7 shows the level of expression of cytokine TNFSF15 inthe group of animals infected and treated with BP-70+ginger+piperine(Group F) compared to the control group of animals that were infectedbut not treated (Group B). Treatment with BP-70+ginger+piperine (GroupF) caused underexpression of TNFSF15 compared to the control group(Group B).

In accordance with the results shown in the present example, treatmentwith sodium butyrate+essential oils, exemplified in the presentinvention by treatment with BP-70+ginger+piperine, was able to show asignificant modulator action on the immune response, causing an increasein the expression of genes coding for the cytokines IL-1β, IL-2 andCD3γδ and a decrease in the expression of the gene coding for thecytokine TNFSF15, giving rise to a protector effect against artificialinfection caused by C. perfringens A.

Furthermore, as demonstrated in the results shown in Example 1,treatment with the additive described in the present invention is alsoable to induce an increase in weight gain of animals subjected to thistreatment and reduce the severity of histological lesions shown by theseanimals as a consequence of infection with C. perfringens A.

The invention claimed is:
 1. A method for promoting animal growthcomprising administering an additive to an animal, wherein said additivecomprises a combination of sodium butyrate with essential oilsconsisting of essential oils of ginger and essential oil piperine,wherein the combination is partially coated with a palm stearin coating.2. The method according to claim 1, wherein the sodium butyrate is in aproportion of 30-70% of the wet weight of the additive.
 3. The methodaccording to claim 1, wherein the sodium butyrate is in a proportion of60% of the wet weight of the additive.
 4. The method according to claim1, wherein the essential oils consisting of essential oils of ginger andessential oil piperine are in a concentration of between 1-20% of thewet weight of the additive.
 5. The method according to claim 1, whereinthe essential oils consisting of essential oils of ginger and essentialoil piperine are in a concentration of 10% of the wet weight of theadditive.
 6. The method according to claim 5, wherein the concentrationof the essential oils of ginger is 5% wet weight of the additive and theconcentration of the essential oil piperine is 5% wet weight of theadditive.
 7. The method according to claim 1, wherein the palm stearincoating is between 30-60% wet weight of the additive.
 8. The methodaccording to claim 1, wherein the palm stearin coating is 30% wet weightof the additive.
 9. The method according to claim 1, wherein theconcentration of sodium butyrate is 60% of the wet weight of theadditive, the concentration of the essential oils consisting ofessential oils of ginger and the essential oil piperine are 10% of thewet weight of the additive, and wherein the concentration of the palmstearin coating is 30% of the wet weight of the additive.
 10. The methodaccording to claim 9, wherein the concentration of the essential oils ofginger is 5% of the wet weight of the additive and the concentration ofthe essential oil piperine is 5% of the wet weight of the additive. 11.The method of claim 1, wherein the animal is selected from the groupconsisting of a chicken, a rabbit, and a pig.